Inflammatory osteolysis, as occurs in rheumatoid arthritis and orthopedic implant loosening, reflects accelerated osteoclast (OC) recruitment and activation. Hence, discovering the means by which local inflammation recruits and activates OCs is central to preventing this crippling complication. Our overriding goal has been to detail the mechanisms by which cytokines, such as TNF, RANKL and M-CSF mediate bone loss. We have achieved the aims of our previous application by characterizing the contributions of various TNF target cells to the osteoclastogenic process and detailing many of the intracellular events by which TNF promotes osteoclastogenesis. In the present proposal we turn to the cytokine essential for all pathological bone resorption, namely RANKL, whose crystal structure, in complex with its receptor, RANK, we resolved in the current funding period. While RANKL is essential for OC differentiation, it also enhances the bone resorptive activity of the mature OC, a process we find depends upon the cytokine's regulation of the cytoskeletal adaptor protein, paxillin and the small GTPase, Rac. Furthermore, our resolution of the RANKL/RANK crystal structure and that of RANKL with the cysteine-rich domains of OPG, the decoy receptor, positions us to develop structure-based RANKL antagonists to arrest RANK signaling in the OC. Given that inhibition of the RANK signaling pathway has proven therapeutic benefit, these novel RANKL inhibitors may impact the treatment of pathological osteolysis. Thus, we hypothesize that: 1) RANKL activation of the OC is mediated via paxillin 2) RANKL activation of the OC is mediated via Rac and 3) Structure-based RANKL antagonists will arrest OC development and function. Our Specific Aims are therefore to 1) Determine the mechanisms by which paxillin mediates RANKL activation of the OC 2) Determine the mechanisms by which RANKL activates Rac in the OC and 3) Engineer variants of RANKL and OPG that disrupt RANK signaling in the OC. PUBLIC HEALTH RELEVANCE. Osteoclasts are the cells which destroy bone, and thus, their increased activity is responsible for most forms of pathological bone loss such as osteoporosis or that attending rheumatoid arthritis. The key molecule which activates ostoclasts is known as RANK ligand (RANKL). The purpose of this proposal is to gain insight into the mechanism by which RANKL activates osteoclasts and to design candidate drugs which will inhibit this event and consequently, prevent pathological bone loss.